Molecular Cell Biology Prof. D. Karunagaran Department of Biotechnology Indian Institute of Technology Madras Module 7 Cell Signaling Mechanisms Lecture 2 GPCR Signaling
Receptors - G protein coupled receptors (GPCRs) conserved in many species. Seven transmembrane (7TM)-receptors. Also called serpentine receptors (Like a snake). Coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) Heterotrimeric - refers to three different proteins Gα, Gβ and Gγ There are also some monomeric G proteins such as Ras (H-Ras, N-Ras and K-Ras) that do not couple with GPCRs GPCR signalling contains many druggable targets There are over 800 GPCRs in the human genome. About 150 of these GPCRs have no known ligands orphan receptors. More than 300 can potentially serve as targets for various disorders 50 60% of all current therapeutic agents are targeting GPCRs directly or indirectly Structure of a typical GPCR All GPCRs contain seven transmembrane alpha-helical Domains, each composed of 25 35 amino acid residues. The highly hydrophobic helices three extracellular and three intracellular loops, amino (N-) terminus extracellular carboxyl (C-) terminus intracellular
Classification of seven transmembrane receptors Class Ligands Class A Rhodopsin family Ligand anchors between the gaps of transmembrane units Class B Secretin family Binding of ligand occurs at the N- terminus (60-80 residues) Class C Glutamate and GABA family Ligand gets trapped between two distinct lobes at the N-terminus Frizzled class Adhesion family Rhodopsin/cone opsin/melanopsin Epinephrine/nor epinephrine Acetyl choline Histamine Serotonin Thromboxane Prostaglandin Melatonin Secretin Glucagon PTH GnRH CRF Glutamate GABA Taste stimuli (sweet) Wnt Hedgehog Taste stimuli (bitter) Chondroitin sulfate Unidentified signals Gonadotrophin releasing hormone GnRH; Corticotropin release factor CRF neuropeptide.
Classifying the ligands Biogenic amines Noradrenaline, dopamine 5-HT, histamine, Acetylcholine Amino acids and ions Glutamate, calcium GABA Lipids LPA, SIP, Prostaglandins, leukotrienes Peptides and proteins Chemokines, angiotensin, thrombin, endothelin, bradykinin Others Light, odorants, nucleotides Class Second messenger G s Β-adrenergic receptor, glucagon receptors, serotonin receptors G i α1-adrenergic receptor G q α2-adrenergic receptor camp Adenylyl cyclase activation camp Adenylyl cyclase inhibition IP3, DAG Phospholipase C activation G 12 LPA receptor RhoGEF mediated activation of Rho
G proteins Numerous members - 16 Gα subunits, five Gβ subunits, and 14 Gγ subunits Most if not all have GTPase activity. The Ga subunits are 39 52 kda proteins divided into four families based on their sequence similarity: Gi/o, Gs, Gq/11, and G12/13. Beta subunits 43kDa; Gamma subunits 7.5-10 kda. Each Gα subunit consists of two domains, a GTPase domain and an alpha helical domain. In between these two domains is a cleft where the guanine nucleotide binds Lipid modification of a Cys residue near the N-terminus of most Gα subunits allows for binding to membrane. C-terminus of all Gα subunits appears to be important for interaction with the receptor. Gβγ is needed for the binding of the Gα subunit to the receptor, for the formation of high-affinity agonist binding, and for receptor catalyzed activation of G protein Gβγ is also attached to the membrane by a lipid modification of the Gg subunit and together the Gβγ dimer acts as a scaffolding protein. Following receptor activation, both Gα and Gβγ act as signaling molecules. Activation of a GPCR results in the release of GDP from Gα. Until GTP binds, a high-affinity complex is formed between the receptor and G protein. The exchange of GDP for GTP leads to dissociation of the Gβγ dimer from the Gα subunit, and both can initiate their own intracellular signaling responses A single GPCR can couple to one or more of Gα proteins G-proteins and second messengers G proteins Activation of Gq may lead to activation of phospholipase C, an enzyme that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3).
IP3 interacts with receptors on intracellular calcium stores resulting in cytosolic release of calcium while DAG can activate protein kinase C isoforms. Other G proteins modulate a large number of second messenger systems including cyclic AMP, cyclic GMP, calmodulin, and kinases.
Adenylyl cyclase
Proteins Regulated by camp-development Phosphorylation (by PKA) Glycogen synthase Phosphorylase b kinase α subunit β subunit Pyruvate kinase (rat liver) Pyruvate dehydrogenase complex (type L) Hormone sensitive lipase Phosphofructokinase-2/fructose 2,6 bisphosphatase Tyrosine hydroxylase Histone H1 Histone H2B Pathway / process regulated Glycogen synthesis Glycogen breakdown Glycolysis Pyruvate to acetyl-coa Triacylglycerol mobilization and fatty acid oxidation Glycolysis / gluconeogenesis Synthesis of L-dopa, dopamine, Norepinephrine and epinephrine DNA condensation DNA condensation
Ligand induced activation of signal-transducing G proteins associated with GPCRs The Gβγ dimer acts as a molecular switch that can be turned on and off via the GTPase cycle
Desensitization Ligand mediated activation of GPCRs is terminated by G protein-coupled receptor kinases (GRKs) GRKs phosphorylate the receptor This results in a simultaneous association with arrestins This complex now interacts with clathrin and the clathrin adaptor AP2 This drives receptor internalization into endosomes. This is a functional process of receptor desensitization. Association of arrestins to activated GPCRs can also lead to the initiation of distinct arrestin-mediated signaling pathways GPCRs may then be taken to lysosomes, where they are ultimately degraded, or to recycling endosomes for recycling back to the cell surface (resensitization).
GPCR Signaling can Generate Multiple Circuits A simple way is to recruit different G proteins from a single ligand-receptor interaction Non-G protein-signaling is also known to occur - G protein-independent signaling GPCR may associate with numerous membrane and intracellular proteins that will potentially alter ligand affinity, ligand selectivity, signaling, cytoskeletal and extracellular matrix interactions, and receptor internalization. GPCRs may also form homo- or heterooligomerization to induce transactivation of other receptors or lead to signal modification. GPCR phosphorylation, acetylation, palmityolation, ubiquitinylation, and myristoylation also modify receptor functional properties. Clearly, all these can generate numerous circuits which have to be integrated. Study Questions 1. What are GPCRs? 2. Wrie a note on the second messengers involved in GPCR signaling 3. Most of the G-proteins possess a) No enzyme activity b) Cyclase activity c) ATPase activity d) GTPase activity 4. Match the following Desensitisation AP2 LPA PKC Clathrin Rho DAG GRKs 5. Receptors without a known ligand are called ---------------